Allergic asthma is the result of airway sensitization and subsequent challenge with allergen, which induces a reduction in gas exchange at the alveoli. Contributing to this impaired gas exchange there is a marked proliferation of airway smooth muscle cells (ASMCs) that also become hypersensitive to both specific (allergen) and nonspecific (acetylcholine) challenge. Given that increases in the intracellular Ca2+ concentration ([Ca2+]i) are integral to airway smooth muscle (ASM) contraction, the central hypothesis is that alterations in Ca2+ signaling are a component of asthma-induced hypersensitivity of ASM. A number of reports indicate that inflammatory agents cause [Ca2+]I to increase in ASMCs. However, the interaction between inflammatory mediators and/or allergic cytokines and increases in ASM cytosolic Ca2+ is unresolved. The two specific aims outlined in the proposal are designed to examine the interaction between inflammatory stimulation, asthma, and cytosolic Ca2+. Specific Aim 1 tests the hypothesis that allergen sensitization and challenge induced ASMC hypersensitivity is due to enhanced intracellular Ca2+ release and extracellular Ca2+ entry. [Ca2+]i will be measured in isolated ASMCs from control and ovalbumin allergen sensitized and challenged Brown-Norway rats, which mimics allergen induced asthma in humans using global Ca2+ imaging and real-time laser scanning confocal microscopy techniques. Changes in basal [Ca2+]i, Kd of Ca2+ increases, change in the spatial and temporal aspects of Ca2+ signaling, or changes in the rate and routes of Ca2+ entry and cytosolic Ca2+ removal with serotonin exposure will be determined. Specific Aim 2 tests the hypothesis that allergen sensitization and challenge induced ASM hypersensitivity is the result of a change in expression of gene products that directly alter Ca2+ signaling. Quantitative RT-PCR and immunohistochemistry techniques will be used to determine if allergen sensitization and challenge changes the expression of non-selective cation channels as well as changes the expression and spatial location of ryanodine and IP3 receptors. Experimental results from these two aims will provide critical pilot data towards the long-term objectives that are to understand the role of changes in Ca2+ signaling to asthma induced hypersensitivity and to elucidate the cell signaling pathways that lead to airway hyperresponsiveness.